sj-pdf-1-ajs-10.1177_03635465231195850 – Supplemental material for Therapeutic Myogenesis Induced by Ultrasound Exposure in a Volumetric Skeletal Muscle Loss Injury Model

Supplemental material, sj-pdf-1-ajs-10.1177_03635465231195850 for Therapeutic Myogenesis Induced by Ultrasound Exposure in a Volumetric Skeletal Muscle Loss Injury Model by Farina Mohamad Yusoff, Ayumu Nakashima, Masato Kajikawa, Shinji Kishimoto, Tatsuya Maruhashi and Yukihito Higashi in The American Journal of Sports Medicine</p

Body Weight, Systemic Hemodynamics and Heart Weight/Tibial Length in Control, Ang II-infused, SNP-infused, and A combined Ang II and SNP-infused Mice.

<p>Ang II indicates angiotensin II; SNP, sodium nitroprusside.</p><p>*P<0.05 vs. before,</p>†<p>P<0.05 vs. control.</p><p>All results are presented as mean±SD.</p><p>Body Weight, Systemic Hemodynamics and Heart Weight/Tibial Length in Control, Ang II-infused, SNP-infused, and A combined Ang II and SNP-infused Mice.</p

Effect of SNP on cGMP accumulation and cell migration in murine and human aortic VSMCs.

<p><b>(A)</b> cGMP accumulation was evaluated after treatment with a vehicle or SNP (40 µmol/L) for 5 minutes in murine VSMCs. n = 6 to 8 in each group. <b>(B)</b> Murine VSMCs migration was evaluated using a modified Boyden chamber assay after 24 hours of incubation with a vehicle or SNP (40 µmol/L) and/or Ang II (40 µmol/L). n = 12 to 13 in each group. <b>(C)</b> cGMP accumulation was evaluated after treatment with a vehicle or SNP (40 µmol/L) for 5 minutes in human aortic VSMCs. n = 5 to 7 in each group. <b>(D)</b> Human aortic VSMCs migration was evaluated using a modified Boyden chamber assay after 24 hours of incubation with a vehicle or SNP (40 µmol/L) and/or Ang II (40 µmol/L). n = 12 in each group. *P<0.05 compared to control; ^§P<0.0001 compared to control; ^§§P<0.0001 compared to Ang II. **P<0.01 compared to control.</p

Effect of SNP on Ang II-induced ROCK activation in human aortic VSMCs.

<p>Human aortic VSMCs were treated with a vehicle or SNP (40 µmol/L) for 3 hours and stimulated with saline or Ang II (40 µmol/L) for the last 1 hour. <b>(A)</b> Representative results of Western blot analysis for p-MBS, t-MBS, ROCK1, ROCK2, and actin. <b>(B)</b> Quantitative analysis of relative ROCK activity (p-MBS/t-MBS). <b>(C)</b> Quantitative analysis of relative ROCK1 expression. <b>(D)</b> Quantitative analysis of relative ROCK2 expression. n = 4 in each group. *P<0.05 compared to control; ^††P<0.01 compared to Ang II. ^†P<0.05 compared to Ang II.</p

Effect of SNP on Ang II-induced ROCK activation in mouse aortas.

<p>Mice were treated with saline or Ang II via implanted micro-osmotic pump infusion (2 mg/kg/day) and treated with saline or SNP via subcutaneous injection (5 mg/kg once a day) for 14 days. Increased blood pressures in Ang II-treated mice were normalized to levels similar to those in mice without Ang II treatment by hydralazine included in drinking water (20 mg/kg/day). <b>(A)</b> Representative results of Western blot analysis for p-MBS, t-MBS, ROCK1, ROCK2, and actin. <b>(B)</b> Quantitative analysis of relative ROCK activity (p-MBS/t-MBS). <b>(C)</b> Quantitative analysis of relative ROCK1 expression. <b>(D)</b> Quantitative analysis of relative ROCK2 expression. n = 5–11 in each group; **P<0.01 compared to control; ^††P<0.01 compared to Ang II.</p

Angiographic Score after Implantation of MSCs.

<p>(A and B) Representative angiography at 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia, (C) Angiographic score at 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia. *P<0.05 vs. the control group.</p

LDPI after Implantation of MSCs.

<p>(A and B) Representative laser LDPIs before and at 1, 7 and 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia, (C) High perfusion is indicated by red, and low perfusion is indicated by blue. Time-dependent change in LDPI index at 1, 7 and 28 days after MSC implantation or saline injection (control).</p

Histological Determination of Capillary Density after Implantation of MSCs.

<p>(A and B) Representative alkaline phosphatase-stained cells at 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia. (Original magnification: ×200), (C and D) Capillary density score and capillary index at 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia. *P<0.05 vs. the control group.</p

Differentiation of Implanted MSCs into Endothelial Cells.

<p>(A and E) Representative CD31-stained cells as red color, (B, F) EGFP-transfected cells as green color, (C and G) DAPI-labeled cell nuclei as blue color, (D and H) CD31/EGFP-double-positive cells as yellow color at 28 days after MSC implantation in most of the ischemic limb tissues (top) and the severely injured areas (bottom) in rabbits with limb ischemia.</p

MSC culture and the ability of adipogenic and osteogenic differentiation.

<p>(A and B) Morphological appearance of rabbit MSC, (A) At day 5 after initial plating, adherent cells were spindle-shaped, (B) At day 10 after initial plating, cells had grown to semiconfluence (Original magnification: ×100), (C) Line graph shows the relationship between cumulative number of cells and days after MSC plating (n = 5), (D) Adipogenic differentiation was confirmed by staining with Oil Red-O to visualize neutral lipid accumulation (Original magnification: ×20), (E) Osteogenic differentiation was confirmed by staining with Alizarin Red S (Original magnification: ×40).</p